Methods for determining and inhibiting rheumatoid arthritis associated with the BRAF oncogene in a subject

ABSTRACT

The invention provides methods for determining whether a subject is suffering from a rheumatoid arthritis associated with the BRAF oncogene comprising contacting isolated fibroblasts from the subject with a molecule or pool of molecules directed to the BRAF oncogene; and culturing the sample in the presence of the agent and determining whether BRAF oncogene expression by the cell is decreased and/or whether cells in the sample return to a less transformed phenotype, exhibit decreased cell proliferation and/or exhibit increased contact inhibition, any of which is indicative that the subject is suffering from a rheumatoid arthritis associated with the BRAF oncogene.

This patent application is a continuation application of U.S. Ser. No.13/440,942, filed Apr. 5, 2012, which claimed the benefit of the filingdate of U.S. Ser. No. 61/472,119, filed Apr. 5, 2011, the contents ofall of which are herein incorporated by reference in their entiretiesinto the present patent application.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Synovial fibroblasts destroy articular cartilage and bone in rheumatoidarthritis, but the mechanism of fibroblast transformation remainselusive. Since gain-of-function mutations of BRAF can transformfibroblasts, we examined BRAF in rheumatoid synovial fibroblasts. Thestrong gain-of-function mutation, V600R, of BRAF found in melanomas andother cancers was identified in first passage synovial fibroblasts fromtwo of nine RA patients and confirmed by restriction site mapping.BRAF-specific siRNA inhibited proliferation of synovial fibroblasts withV600R mutations. A BRAF aberrant splice variant with an intact kinasedomain and partial loss of the N-terminal autoinhibitory domain wasidentified in fibroblasts from an additional patient, and fibroblastproliferation was inhibited by BRAF-specific siRNA. Our finding is thefirst to establish mechanisms for fibroblast transformation responsiblefor destruction of articular cartilage and bone in rheumatoid arthritisand establishes a new target for therapeutic intervention.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that occursin 1% of the population and is characterized by progressive erosivearthritis of multiple joints associated with increased mortality.Although the inflammatory reaction contains numerous cell types,synovial fibroblasts have been identified as the cell responsible forinvasion and destruction of cartilage and bone (Muller-Ladner, U., etal. (1996) Am J Pathol, 149, 1607-1615; Pap, T., et al. (2000) ArthritisRes, 2, 361-367; Buckley, C. D., et al. (2001) Trends Immunol, 22,199-204; Muller-Ladner, U., et al. (2005) Nat Clin Pract Rheumatol, 1,102-110; Pap, T., et al. (2005) Ann Rheum Dis, 64 Suppl 4, iv52-54;Karouzakis, E., et al. (2006) Immunol Lett, 106, 8-13; Huber, L. C., etal. (2006) Rheumatology (Oxford), 45, 669-675). Rheumatoid synovialfibroblasts show evidence of transformation indicated by excessiveproliferation, loss of contact inhibition, and increased migration (Pap,T., et al. (2000) Arthritis Res, 2, 361-367; Karouzakis, E., et al.(2006) Immunol Lett, 106, 8-13; Mercer, K., et al. (2005) Cancer Res,65, 11493-11500). Transformation of RA synovial fibroblasts has alsobeen demonstrated in an animal model of RA employing xenograft implantsof RA synovium as evidenced by metastasis of implanted synovialfibroblasts with localization and binding to cartilage (Lefevre, S., etal. (2009) Nat Med, 15, 1414-1420). The mechanism of RA synovialfibroblast transformation has not been identified, but it is criticalfor the rational design of therapies to prevent joint destruction. Inspite of evidence for neoplastic transformation of RA synovialfibroblasts, oncogenes potentially responsible for transformation havenot been identified. Since gain-of-function mutations in the BRAFoncogene have been shown to transform embryonic fibroblasts but notseveral other somatic cell types, we examined rheumatoid synovialfibroblasts for the presence of BRAF mutations (Mercer, K., et al.(2005) Cancer Res, 65, 11493-11500).

SUMMARY OF THE INVENTION

The invention provides methods for determining or diagnosing whether asubject is suffering from a rheumatoid arthritis (RA) associated withthe BRAF oncogene. In one embodiment, the method comprises contactingisolated fibroblasts from the subject with a molecule or pool ofmolecules directed to the BRAF oncogene. The method further comprisesdetecting association of the agent with the BRAF oncogene, e.g. suchthat BRAF oncogene expression is decreased and fibroblasts return to aless transformed phenotype, decreased cell proliferation and increasedcontact inhibition, being indicative that the subject is suffering froma rheumatoid arthritis associated with the BRAF oncogene.

The invention additionally provides methods for inhibiting cells havinga mutant BRAF associated with RA and methods for treating subjectshaving RA associated with the BRAF oncogene.

The invention further provides methods for identifying molecules thatbind or block the BRAF protein in a rheumatoid arthritis cell. Themethod comprises contacting a molecule of interest with the BRAFprotein. The method also comprises determining whether the molecule ofinterest alters BRAF protein activity, alteration of the BRAF proteinactivity being indicative that the molecule of interest binds or blocksthe BRAF protein in the cell.

Additionally provided are compositions containing one or more agentsthat bind or block BRAF for inhibiting RA.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. PCR amplification of BRAF from RA fibroblasts. FIG. 1A.Loss of fibroblast contact inhibition. Rheumatoid synovial fibroblastswere grown in T25 flasks and observed microscopically. Fibroblasttransformation is suggested by the loss of contact inhibition. Arepresentative example is shown. Bar, 20 μm. FIG. 1B. BRAF amplified byRT and nested PCR. Nested PCR amplified a DNA fragment of about 2300 bpin all patients. DNA sequencing confirmed the presence of BRAF. Aberrantsplice variants were observed in RA6, RA8, and RA9 as confirmed by DNAsequencing. FIG. 1C. Splice variants of BRAF from rheumatoid synovialfibroblasts. Shown are the deletion of exons in splice variants fromRA6, RA8, and RA9. RA8 has a splice variant that deletes a portion ofthe BRAF autoinhibitory domain but retains the kinase domain.

FIG. 2. Mutations of BRAF from rheumatoid synovial fibroblasts. V600Rwas identified in BRAF from RA synovial fibroblasts in 2 of 9 RApatients. The first two panels show chromatograms from RA1 and RA6consistent with two separate nucleotide sequences encoding two differentamino acids at residue 600, wild-type and V600R mutant. Panel 3 is achromatogram from patient RA3 showing the wild-type nucleotide sequenceencoding valine at residue 600.

FIGS. 3A-3C. Restriction enzyme mapping. FIG. 3A. The SfcI restrictionsite includes the first nucleotide of the codon for BRAF residue 600.FIG. 3B. Incomplete SfcI digestion of 2300 bp BRAF cDNA obtained by PCR(inverted image) is consistent with V600R of BRAF from RA6. In contrast,2300 bp BRAF cDNA from RA3 without BRAF mutation shows completedigestion by SfcI. FIG. 3C. Incomplete SfcI digestion of 191 bp BRAFcDNA fragment obtain by “Fast COLD PCR” (inverted image) is consistentwith V600R of BRAF from RA1. In contrast, 191 bp BRAF cDNA from RA3without BRAF mutation shows complete digestion by SfcI. Arrows indicateuncleaved cDNA following digestion with excess SfcI.

FIGS. 4A-4B. Inhibition of RA fibroblast growth by BRAF siRNA. FIG. 4A.Synovial fibroblasts from 5 RA patients and 1 OA patient were culturedfor 72 hours in the presence of control and BRAF-specific siRNA.Significant inhibition of growth in response to BRAF siRNA was observedin fibroblasts from RA1 and RA6 with a V600R mutation of BRAF and in RA8with a BRAF splice variant containing a partial deletion of theautoinhibitory domain. FIG. 4B. BRAF siRNA inhibits production of BRAF.Synovial fibroblasts from RA6 were incubated with BRAF siRNA for 72hours and assessed for BRAF production by Western blot. Actin served asa loading control:

FIGS. 5A-5B. BRAF Aberrant Splice Variants in Rheumatoid ArthritisSynovial Fibroblasts. FIG. 5A. Synovial tissue was obtained frompatients with rheumatoid arthritis undergoing joint-replacement surgery.Synovial tissue was treated with collagenase, and cell suspensions werecultured in DMEM/F12 (1:1) medium containing 10% FCS. RNA was isolatedfrom pure fibroblast cultures, and cDNA was produced with BRAF-specificprimers outside of the coding sequence by RT-PCR. Nested PCR wasperformed with BRAF-specific primers within the coding sequence.Aberrant splice variants were identified by direct nucleotide sequencingof gel-purified DNA. FIG. 5B. Direct sequencing of gel-purified DNAshowed aberrant BRAF splice variants characterized by exon skipping.Shown are deleted exons of BRAF.

FIG. 6. RNA was isolated from rheumatoid synovial fibroblasts, and KRAScDNA was obtained by RT-PCR with KRAS-specific primers located in thenon-coding sequence. Nested PCR was then performed with KRAS-specificprimers located within the coding sequence. PCR fragments were thenligated into PCR2.1 for DNA sequencing. KRAS mutants were identified insynovial fibroblasts from 7 of 9 patients.

FIG. 7. COS-7 cells transfected with an expression vector containingKRAS mutants in order to determine whether KRAS mutations wereresponsible for constitutive MAPK activation. Mutant G12D served as apositive control. Mutants T74A and E31Q constitutively activated MAPKactivity.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety.

The term “BRAF oncogene” includes nucleic acid sequences containing oneor more additions, deletions or substitutions relative to the BRAFoncogene described herein, e.g., SEQ ID NO:1 and BRAF oncogenes fromhumans and other species. In addition, the term “BRAF oncogene” includesRNA transcripts containing all or a portion of the nucleic acid sequencedescribed in SEQ ID NO:1, as well as the genomic sequences correspondingto the SEQ ID NO:1 along with its transcripts and the associatedintrons, exons, untranslated leader region, splice donors, spliceacceptors, polyadenylation signals, promoter, and transcriptionalenhancers, as may be found in publically available sequence,bibliological, and journal article databases, such as but not limited tothose housed at the National Center for Biotechnology Information,Bethesda, Md. (web address: www.ncbi.nlm.nih.gov) and HUGO GeneNomenclature Committee, Cambridge, UK (http://www.genenames.org) andtheir associated web links. Another name for “BRAF” is “v-raf murinesarcoma viral oncogene homolog B 1.”

The term “BRAF protein” includes proteins encoded by the nucleic acidsequence provided in SEQ ID NO:1 and may contain one or more additions,deletions or substitutions relative to the wild-type as given in SEQ IDNO:2 and includes the variants described herein. Furthermore, the term“BRAF protein” includes BRAF proteins from humans and other species.

The phrase “mutant or variant BRAF or aberrant BRAF splice variants”refers to a BRAF oncogene that can be processed or spliced, by one ormore processing reactions, to produce mutant or variant BRAF or aberrantBRAF splice variants. BRAF variants may generally contain either adeletion (part or whole) of a RAS-binding domain (RBD) at exons 3-5(including anywhere between exons 3-5) or a deletion (partial or whole)of the RBD and the kinase domain (e.g., at exons 11-17 (including anychange that would eliminate the kinase activity of the oncogene)).Additionally, the change may include mutations that results in BRAFproteins that exhibit gain-of-function. The gain-of-function mutationincludes point mutations such as that affecting valine at amino acid 600or deletions of the BRAF protein.

The source of BRAF can be a live organism (including a human patient, ora laboratory or veterinary animal, such as dog, pig, cow, horse, rat ormice), a sample therefrom (such as a tissue or body fluid, or extractthereof), a cell (such as a primary cell or cell line, or extractthereof), extracellular medium or matrix or milieu, or isolated protein.

The phrase “inhibit” or “inhibiting” with respect to cells (e.g.fibroblasts) associated with rheumatoid arthritis (RA) having the BRAFoncogene, refers to any one or more of a detectable inhibition of thegrowth of cells (e.g. fibroblasts) (in or from the subject); BRAFoncogene expression is increased; cells (e.g. fibroblasts) return to amore transformed phenotype; and decreased contact inhibition by thecells (e.g. fibroblasts). Inhibiting cells (e.g. fibroblasts) associatedwith RA having the BRAF oncogene can be evidenced, for example, by adecrease of at least 5%, such as at least 10%, 20%, 30%, 40%, 50%, 75%,90% or more, of the number, or relative number, of cells (e.g.fibroblasts), relative to a reference level. Inhibition can be adecrease that is a statistically significant difference relative to thereference level.

The phrase “contacting or exposing” refers to bringing into association,either directly or indirectly, two or more substances. Contacting mayoccur in vivo, ex vivo or in vitro. For example, a source of BRAF e.g.,a cell or tissue, that is a human or other animal may be contacted withan agent (e.g., a molecule (such as nucleic acid molecules), compound orantibody including fragments thereof), for example, by therapeutic orprophylactic administration of the agent. A source of BRAF that is afluid, such as extracellular medium, can be contacted with an agent, forexample, by admixing the agent with the fluid.

The phrase “treating” or “treatment” refers to any manner in which oneor more of the symptoms of a disease or disorder (such as RA) areameliorated or otherwise beneficially altered, whether in a permanent ortemporary manner, which can be attributed to or associated withadministration of the agent or composition herein. The term encompassesany pharmaceutical use, including prophylactic uses in which thedevelopment of one or more of the symptoms of a disease or disorder(e.g., RA) is prevented, delayed or reduced, whether in a permanent ortemporary manner, which can be attributed to or associated withadministration of the composition.

Methods of the Invention

The present invention provides methods for determining or diagnosingwhether a subject is suffering from a rheumatoid arthritis associatedwith the BRAF oncogene.

In an embodiment of the invention, the method comprising contacting asample from the subject with an agent (e.g. a molecule or pool ofmolecules) directed to the BRAF oncogene or its transcript(s). Themethod further comprises detecting association of the agent with theBRAF oncogene or its transcript(s) (e.g., by detecting a complex betweenthe BRAF oncogene or its transcript(s) and the agent). The associationmay be indicative that the subject is suffering from a rheumatoidarthritis associated with the BRAF oncogene. In an embodiment of theinvention, the BRAF oncogene has a mutation so that the codon encodingvaline at nucleotide position 1798-1800 of SEQ ID NO:1 is changed to anyof the codons CGU, CGC, CGA, CGG, AGA, and AGG which encodes arginine.

In another embodiment, the method comprises contacting a sample from thesubject with an agent or agents directed to different portions of theBRAF oncogene; and detecting the association or lack of association ofthe agent or agents with different portions of the BRAF oncogene.Further, the method provides quantifying the amount of association todifferent portions of the BRAF oncogene. In this embodiment, the changesin the amount of association affecting one or more portion(s) of theBRAF oncogene but not other(s) is indicative that the subject issuffering from a rheumatoid arthritis associated with the BRAF oncogene.Further, in this embodiment, the BRAF oncogene is a mutant BRAFoncogene.

In a further embodiment, the method comprises (a) contacting or exposinga sample (e.g. isolated fibroblasts) from the subject (e.g., a subjectsuffering from rheumatoid arthritis) with an agent (e.g. a molecule orpool of molecules) directed to the BRAF oncogene. Further, the methodcomprising detecting association of the agent with the BRAF oncogene inthe sample such that (1) BRAF oncogene expression is decreased, (2)fibroblasts return to a less transformed phenotype, and/or (3)fibroblasts exhibit decreased cell proliferation and/or increasedcontact inhibition, one or more of these features being indicative thatthe subject is suffering from a rheumatoid arthritis associated with theBRAF oncogene.

In another embodiment, the method comprises contacting a sample from thesubject with an agent (e.g., a molecule or pool of molecules) directedto the BRAF oncogene. Further, the method comprises culturing the samplein the presence of the molecule or pool of molecules and determiningwhether BRAF oncogene expression by the cell is decreased and/or whethercells in the sample return to a less transformed phenotype, exhibitdecreased cell proliferation and/or exhibit increased contactinhibition, any of which is indicative that the subject is sufferingfrom a rheumatoid arthritis associated with the BRAF oncogene.

In accordance with the practice of the invention, the molecule that isdirected to or binds the BRAF oncogene may be a nucleic acid molecule.In one embodiment, the nucleic acid molecule may be a RNA molecule, andthe RNA molecule is a siRNA molecule. Additionally, the nucleic acidmolecule may be an antisense molecule. In a further embodiment, thesiRNA or antisense molecule is directed to the BRAF oncogene sequence.

In an embodiment of the invention, the sample is a cell sample, abiological fluid sample (e.g., synovial fluid) or tissue sample.

In one embodiment, the sample includes fibroblasts, e.g., fibroblastsisolated from synovial tissue or fluid from the subject.

In another embodiment, the BRAF oncogene comprises one or more nucleicacid mutations in nucleotide position 1798 to nucleotide position 1800of SEQ ID NO:1. For example, in one embodiment, the mutation results ina BRAF protein, wherein valine at amino acid position 600 of SEQ ID NO:2is changed to an amino acid other than valine (such as alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, and tyrosine). Ina preferred embodiment, the mutation at amino acid position 600 of SEQID NO:2 is a change from valine to arginine (V600R). The V600R changeinvolves replacing the codon GTG (at nucleotide positions 1798-1800 ofSEQ ID NO:1) which encodes valine with any of the codons CGU, CGC, CGA,CGG, AGA, and AGG which encodes arginine.

In a further embodiment, the BRAF oncogene is analyzed for a mutation ormutations in codon 600 by reverse transcribing RNA isolated from thefibroblasts from synovial tissue of the subject so as to obtain cDNA.The BRAF oncogene may be analyzed for a mutation or mutations in codon600 also by amplifying the cDNA so obtained and determining whether theBRAF oncogene at codon 600 is mutated such that codon 600 valine isreplaced with an amino acid other than valine (e.g. arginine).

In another embodiment, the BRAF oncogene encodes a BRAF protein as shownin SEQ ID NO:2 beginning with methionine at amino position 1 and endingwith histidine at amino acid position 766. In this embodiment, the BRAFoncogene begins with adenosine at nucleotide position 1 and ends withcytosine at nucleotide position 2298 of SEQ ID NO:1.

In an embodiment of the invention, the BRAF oncogene encodes a BRAFprotein having an auto-inhibitory domain which has an amino acidsequence beginning at position 47 through position 245 of SEQ ID NO:1encoded by a nucleic acid sequence beginning with nucleotide position139 and ending with nucleotide position 735 of SEQ ID NO:1,respectively, and a kinase domain which has an amino acid sequencebeginning at position 433 and ending with position 726 of SEQ ID NO:1which is encoded by a nucleic acid sequence beginning with nucleotideposition 1297 and ending with nucleotide position 2178 of SEQ ID NO:1,respectively.

In a further embodiment, the BRAF oncogene encodes a mutant BRAF proteinwherein the auto-inhibitory domain of the BRAF protein is deleted, e.g.,entirely or partially. In one example, the kinase domain is intact orremains unchanged and is functional (i.e., retains its catalyticfunction) and/or is constitutively active, the latter, again-of-function mutation or activating mutation within the kinasedomain. In another example, the kinase domain is changed (e.g., bymutating one of more amino acids within position 433 through position726 of SEQ ID NO:1) but the kinase domain remains functional and/or isconstitutively active, the latter, a gain-of-function mutation oractivating mutation within the kinase domain.

In an additional embodiment, the BRAF protein is a mutant BRAF proteinof SEQ ID NO:1 wherein the auto-inhibitory domain of the BRAF proteinbeginning with amino acid at position 47 and ending with amino acid atposition 245 (which is encoded by nucleic acid sequence beginning atnucleotide position 139 and ending with nucleotide position 735 of SEQID NO:1), is deleted, entirely or partially, but the kinase domainbeginning with amino acid at position 433 and ending with amino acidposition 726 of SEQ ID NO:2 (which is encoded by nucleic acid sequencebeginning at nucleotide position 1297 and ending with nucleotideposition 2178 of SEQ ID NO:1), is intact or may be altered at one ormore nucleic acid or amino acid position but remains functional as akinase domain (e.g., such as that exhibited by the kinase domain ofunaltered BRAF oncogene).

In another embodiment, the BRAF oncogene is a mutant BRAF oncogene (e.g.so that it is an alternatively spliced BRAF transcript as shown in SEQID NO:1) which is missing exons 3-8 beginning with nucleotide position241 through nucleotide position 1140 of SEQ ID NO:1 and encodes a mutant(e.g., truncated) BRAF protein that is missing an amino acid sequencebeginning with amino acid position 81 to amino acid position 380 of SEQID NO:1.

In a further embodiment, the BRAF oncogene is an alternatively splicedBRAF transcript as shown in SEQ ID NO:1 which is missing the nucleicacid sequence beginning at nucleotide position 505 to nucleotideposition 1992 so that it encodes a mutant BRAF protein shown in SEQ IDNO:1 but which is missing an amino acid sequence beginning at amino acidposition 169 through amino acid position 664.

In a yet further embodiment, wherein the BRAF oncogene is analternatively spliced BRAF transcript as shown in SEQ ID NO:1 which ismissing the nucleic acid sequence beginning at nucleotide position 139to nucleotide position 1992 so that it encodes a mutant BRAF proteinshown in SEQ ID NO:1 but which is missing an amino acid sequencebeginning at amino acid position 47 through amino acid position 664.

In a still further embodiment, the BRAF oncogene is an alternativelyspliced BRAF transcript as shown in SEQ ID NO:1 which is missing thenucleic acid sequence beginning at nucleotide position 139 to nucleotideposition 1860 so that it encodes a mutant BRAF protein shown in SEQ IDNO:1 but which is missing an amino acid sequence beginning at amino acidposition 47 through amino acid position 620.

In an additional embodiment, the BRAF oncogene is an alternativelyspliced BRAF transcript as shown in SEQ ID NO:1 which is missing thenucleic acid sequence beginning at nucleotide position 505 to nucleotideposition 1695 so that it encodes a mutant BRAF protein shown in SEQ IDNO:1 but which is missing amino acid sequence beginning at amino acidposition 169 and ending at amino acid position 565.

The invention additionally provides methods for determining whether asubject is suffering from a rheumatoid arthritis which is associatedwith either a mutant BRAF oncogene or an aberrant activation of theRAS-ERK-MAPK signaling pathway or both. In one embodiment, the methodcomprises determining whether the subject is suffering a rheumatoidarthritis associated with a mutant BRAF oncogene by a method describedabove. The method also comprises determining whether the subject issuffering from a aberrant activation of the RAS-ERK-MAPK signalingpathway for example by isolating nucleic acid sequence of each componentof the signaling pathway from the sample; determining the nucleic acidsequence and comparing it to the wild-type nucleic acid sequence toidentify presence of a mutation(s) in the coding region; expressing themutant protein in a mammalian cell so as to be able to determine itsactivity relative to the non-mutant or wild-type protein, similarlyexpressed; and determining the phosphorylation state of differentcomponents of the signaling pathway, such that an increase in thephosphorylation state observed with the mutant protein relative to thenon-mutant or wild-type protein being indicative of a subject sufferingfrom a rheumatoid arthritis associated with aberrant activation of thesignaling pathway.

In accordance with the practice of the invention, a component of thesignaling pathway includes HRAS, NRAS, KRAS, ARAF, BRAF, CRAF, MEK1,MEK2, ERK1 and ERK2 coding sequences.

Further, in accordance with the practice of the invention, the samplemay be a cell sample, a biological fluid sample (e.g., synovial fluid)or tissue sample. The sample contains fibroblasts such as fibroblasts.

In an embodiment of the invention, the coding sequence for the mutant orwild-type protein is expressed in a mammalian cell through the use of amammalian expression system either inducible or constitutively,following introduction of the mammalian expression system along with thecoding sequences of interest into the mammalian cell. Examples ofmammalian cells include COS-7 cell, HEK-293 cell, U2OS cell, and HeLa.

In an embodiment of the invention, the phosphorylation state of HRAS,NRAS, KRAS, ARAF, BRAF, CRAF, MEK1, MEK2, ERK1 or ERK2 protein may bedetermined with a phospho-specific antibody.

The invention additionally provides methods for inhibiting cells havingan activated BRAF oncogene associated with rheumatoid arthritis. In oneembodiment, the method comprises contacting the cells with an agent thatbinds the BRAF oncogene and thereby inhibiting the cells.

The invention additionally provides methods for treating a subjectsuffering from rheumatoid arthritis associated with a BRAF oncogene. Inone embodiment, the method comprises administering an agent moleculethat binds the BRAF protein to the subject suffering from a rheumatoidarthritis associated with the BRAF oncogene and thereby treating thesubject suffering from rheumatoid arthritis.

In accordance with the practice of the invention, the agent may beadministered in or around a joint area of the subject such as in thejoint area of the knee, ankle, elbow, hand, shoulder, or hip.

Also, in one embodiment, the agent that binds the BRAF protein is a BRAFinhibitor. In accordance with the practice of the invention, the BRAFinhibitor may be administered alone or in combination (concurrent orsequential) with one or more medications for rheumatoid arthritis (e.g.,immunosuppression agents).

Examples of immunosuppressive agents include methotrexate,cyclophosphamide, cyclosporine, cyclosporin A, chloroquine,hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts,D-penicillamine, leflunomide, azathioprine, anakinra, infliximab(REMICADE®), etanercept, TNFα blockers, a biological agent that targetsan inflammatory cytokine, and, Nonsteroidal Anti-inflammatory Drugs(NSAIDS). NSAIDs include, but are not limited to acetyl salicylic acid,choline magnesium salicylate, diflunisal, magnesium salicylate,salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen,flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate,naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

Examples of suitable BRAF inhibitors include but are not limited toPLX4032 (also known asN-(3-(5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide)(Plexxikon, Inc., Berkeley, Calif.; (24)), RAF265 (also known as1-methyl-5-(2-(4-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yloxy)-N-(4-(trifluoromethypphenyl)-1H-benzo[d]imidazol-2-amine)(CHIR-265; Novartis Pharmaceuticals, Basel, Switzerland), Sorafenib(also known as4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide)(BAY43-9006; Bayer, Pittsburgh, Pa.), XL281 (Exelixis, San Francisco,Calif.), SB-590885 (also known as5-[2-[4-[2-(Dimethylamino)ethoxy]phenyl]-5-(4-pyridinyl)-1H-imidazol-4-yl]-2,3-dihydro-1H-inden-1-oneoxime)(SmithKline Beecham, Philadelphia, Pa.), and PLX4720 (also known asN-(3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide)(Plexxikon, Inc., Berkeley, Calif.).

In another embodiment, the method for treating a subject suffering fromrheumatoid arthritis associated with a BRAF oncogene comprisesadministering a molecule that targets the BRAF oncogene or transcriptsthereof in the subject and thereby treating the subject suffering fromrheumatoid arthritis.

In accordance with the practice of the invention, the molecule thattargets the BRAF oncogene or transcripts thereof may be a nucleic acidmolecule. For example, the nucleic acid molecule may be a RNA moleculesuch as a siRNA molecule. In one embodiment, the siRNA molecule isdirected to the BRAF oncogene sequence.

In another embodiment, the method for treating a subject suffering fromrheumatoid arthritis associated with the BRAF oncogene comprisesadministering an agent (e.g., a molecule) that inhibits the RAS-RAF-MAPKsignaling pathway to the subject suffering from a rheumatoid arthritisassociated with the BRAF oncogene and thereby treating the subjectsuffering from rheumatoid arthritis.

In an embodiment, the agent that inhibits the RAS-RAF-MAPK signalingpathway is a MEK inhibitor or an ERK inhibitor. Suitable examples of MEKinhibitors include but are not limited to AZD6244 (also known as6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide)(ARRY-142886; AstraZeneca, London, England), PD0325901 (also known as(R)—N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide),CI-1040 (also known as2-(2-Chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide),and XL518 (also known as(S)-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydroxy-3-(piperidin-2-yl)cyclobutyl)methanone)(Exelixis, San Francisco, Calif.).

Suitable examples of ERK inhibitors include but are not limited toFR180204 (also known as5-(2-Phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine)(EMD4Biosciences, Merck KGaA, Darmstadt, Germany), CAY10561 (also knownasN-[1-(3-chloro-4-fluorophenyl)-2-hydroxyethyl]-4-[4-(3-chlorophenyl)-1H-pyrazol-3-yl]-1H-pyrrole-2-carboxamide)(Cayman Chemical, Ann Arbor, Mich.) and 328006 (also known as3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione)(Merck KGaA, Darmstadt, Germany).

The amount of an agent administered to a subject varies depending onseveral factors including the efficacy of the drug on a specific subjectand the toxicity (i.e. the tolerability) of a drug to a specificsubject.

In one embodiment, the agent may be administered to the subject with animmunosuppressive agent (concurrently or sequentially). In anotherembodiment, the agent may be administered before or after administrationof the suppressive agent.

Screening Assays

The invention further provides a method of identifying or screeningmolecules that bind or block the BRAF protein in a rheumatoid arthritiscell comprising contacting a molecule of interest with the BRAF protein,and determining whether the molecule of interest alters BRAF proteinactivity, alteration of the BRAF protein activity being indicative thatthe molecule of interest binds or blocks the BRAF protein in the cell.

Compositions of the Invention

The present invention relates to compositions comprising animmunosuppressive agent and a BRAF inhibitor that bind and/or aredirected to the BRAF oncogene or proteins. In accordance with theinvention, the composition may further comprise an agent that inhibitsthe RAS-RAF-MAPK signaling pathway such as MEK inhibitor or an ERKinhibitor.

In one embodiment, the BRAF inhibitor may be a nucleic acid molecule,e.g. a RNA molecule such as a siRNA molecule.

The invention also relates to BRAF inhibitor molecules or agents of theinvention that bind a portion of a BRAF protein as shown in SEQ ID NO:1beginning with methionine at nucleotide position 1 and ending withhistidine at nucleotide position 2298.

In one embodiment, the molecule or agent may be a protein or polypeptidesuch as an antibody or fragment thereof that binds a BRAF protein. Forexample, an antibody that specifically binds the BRAF protein at itsC-terminus (e.g., the portion encoded by exon 18 as shown in SEQ IDNO:1). Antibodies again BRAF are well known. Examples include LS-C97196,LS-B1627, LS-B3862, LS-B3444, and LS-C49616 (all from Lsbio.com).Antibodies against a BRAF protein at its C-terminus are well known(Clampi et al. J. Clin. Invest. 115:94-101 (2005); Lsbio.com). Theantibody may be polyclonal, monoclonal, chimeric, or humanized. Theantibody may be a full length antibody or may be a fragment of anantibody such as a Fab molecule or F(ab′)₂ molecule. Another antibodyfragment can have as the variable region, an Fv or single chain Fvconfiguration.

In a further embodiment, the agent may bind to a portion of a BRAFprotein wherein the auto-inhibitory domain of the BRAF protein beginningwith nucleotide position 139 at amino acid position 47 and ending withnucleotide position 735 at amino acid position 245 of SEQ ID NO:1 isdeleted, entirely or partially, but the kinase domain beginning withnucleotide position 1297 at amino acid position 433 and ending withnucleotide position 2178 at amino acid position 726 of SEQ ID NO:1 isintact or remains functional.

Examples of immunosuppressive agents include methotrexate,cyclophosphamide, cyclosporine, cyclosporin A, chloroquine,hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts,D-penicillamine, leflunomide, azathioprine, anakinra, infliximab(REMICADE®), etanercept, TNFα blockers, a biological agent that targetsan inflammatory cytokine, and, Nonsteroidal Anti-inflammatory Drugs(NSAIDS). NSAIDs include, but are not limited to acetyl salicylic acid,choline magnesium salicylate, diflunisal, magnesium salicylate,salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen,flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate,naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

Examples of suitable BRAF inhibitors include but are not limited toPLX4032 (also known asN-(3-(5-(4-chlorophenyl)-1′-1-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide)(Plexxikon, Inc., Berkeley, Calif.; (24)), RAF265 (also known as1-methyl-5-(2-(4-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yloxy)-N-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-2-amine)(CHIR-265; Novartis Pharmaceuticals, Basel, Switzerland), Sorafenib(also known as4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide)(BAY43-9006; Bayer, Pittsburgh, Pa.), XL281 (Exelixis, San Francisco,Calif.), SB-590885 (also known as5-[2-[4-[2-(Dimethylamino)ethoxy]phenyl]-5-(4-pyridinyl)-1H-imidazol-4-yl]-2,3-dihydro-1H-inden-1-oneoxime)(SmithKline Beecham, Philadelphia, Pa.), and PLX4720 (also known asN-(3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide)(Plexxikon, Inc., Berkeley, Calif.).

Suitable examples of MEK inhibitors include but are not limited toAZD6244 (also known as6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide)(ARRY-142886; AstraZeneca, London, England), PD0325901 (also known as(R)—N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide),CI-1040 (also known as2-(2-Chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide),and XL518 (also known as(S)-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydroxy-3-(piperidin-2-yl)cyclobutyl)methanone)(Exelixis, San Francisco, Calif.).

Suitable examples of ERK inhibitors include but are not limited toFR180204 (also known as5-(2-Phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine)(EMD4Biosciences, Merck KGaA, Darmstadt, Germany), CAY10561 (also knownasN-[1-(3-chloro-4-fluorophenyl)-2-hydroxyethyl]-4-[4-(3-chlorophenyl)-1H-pyrazol-3-yl]-1H-pyrrole-2-carboxamide)(Cayman Chemical, Ann Arbor, Mich.) and 328006 (also known as3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione)(Merck KGaA, Darmstadt, Germany).

-   -   A. Compositions for Oral Administration

Oral pharmaceutical dosage forms are either solid, gel or liquid. Thesolid dosage forms may be tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tabletswhich may be enteric-coated, sugar-coated or film-coated. Capsules maybe hard or soft gelatin capsules, while granules and powders may beprovided in non-effervescent or effervescent form with the combinationof other ingredients known to those skilled in the art.

-   -   1. Solid Compositions for Oral Administration

In certain embodiments, the compositions are solid dosage forms, in oneembodiment, capsules or tablets. The tablets, pills, capsules, trochesand the like can contain one or more of the following ingredients, orcomponents of the composition of a similar nature: a binder; alubricant; a diluent; a glidant; a disintegrating agent; a coloringagent; a sweetening agent; a flavoring agent; a wetting agent; an emeticcoating; and a film coating. Examples of binders includemicrocrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, molasses, polyinylpyrrolidine, povidone,crospovidones, sucrose and starch paste. Lubricants include talc,starch, magnesium or calcium stearate, lycopodium and stearic acid.Diluents include, for example, lactose, sucrose, starch, kaolin, salt,mannitol and dicalcium phosphate. Glidants include, but are not limitedto, colloidal silicon dioxide. Disintegrating agents includecrosscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Coloring agents include, for example, any of theapproved certified water soluble FD and C dyes, mixtures thereof; andwater insoluble FD and C dyes suspended on alumina hydrate. Sweeteningagents include sucrose, lactose, mannitol and artificial sweeteningagents such as saccharin, and any number of spray dried flavors.Flavoring agents include natural flavors extracted from plants such asfruits and synthetic blends of compounds which produce a pleasantsensation, such as, but not limited to peppermint and methyl salicylate.Wetting agents include propylene glycol monostearate, sorbitanmonooleate, diethylene glycol monolaurate and polyoxyethylene lauralether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammuoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

The components of the composition could be provided in a form thatprotects it from the acidic environment of the stomach. For example, thecomposition can be formulated in an enteric coating that maintains itsintegrity in the stomach and releases the active components of thecomposition in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The composition can also be administeredas a component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup may contain, in addition to the activecomponents of the composition, sucrose as a sweetening agent and certainpreservatives, dyes and colorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H2 blockers, and diuretics. The activeingredient is an agent or pharmaceutically acceptable derivative thereofas described herein. Higher concentrations, up to about 98% by weight ofthe active ingredient may be included.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

-   -   2. Liquid Compositions for Oral Administration

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous-liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicacid, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Sweetening agents include sucrose, syrups, glycerin andartificial sweetening agents such as saccharin. Wetting agents includepropylene glycol monostearate, sorbitan monooleate, diethylene glycolmonolaurate and polyoxyethylene lauryl ether. Organic acids includecitric and tartaric acid. Sources of carbon dioxide include sodiumbicarbonate and sodium carbonate. Coloring agents include any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include natural flavors extracted from plants suchfruits, and synthetic blends of compounds which produce a pleasant tastesensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is in oneembodiment encapsulated in a gelatin capsule. For a liquid dosage form,the solution, e.g., for example, in a polyethylene glycol, may bediluted with a sufficient quantity of a pharmaceutically acceptableliquid carrier, e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active components of the composition orsalt in vegetable oils, glycols, triglycerides, propylene glycol esters(e.g., propylene carbonate) and other such carriers, and encapsulatingthese solutions or suspensions in hard or soft gelatin capsule shells.Briefly, such formulations include, but are not limited to, thosecontaining components of the composition provided herein, a dialkylatedmono- or poly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

-   -   B. Injectables, Solutions and Emulsions

Parenteral administration, in one embodiment characterized by injection,either subcutaneously, intramuscularly or intravenously is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained is also contemplated herein.Briefly, components of the composition provided herein may be dispersede.g., in a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The components of the composition diffuse through the outerpolymeric membrane in a release rate controlling step. The percentage ofactive components of the composition is highly dependent on the specificnature thereof, as well as the activity of the components of thecomposition and the needs of the subject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcelluose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN™ 80). A sequestering or chelatingagent of metal ions includes EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles; and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the pharmaceutically active components of thecomposition is adjusted so that an injection provides an effectiveamount to produce the desired pharmacological effect. The exact dosedepends on the age, weight and condition of the patient or animal as isknown in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing active components of the composition is aneffective mode of administration. Another embodiment is a sterileaqueous or oily solution or suspension containing an active materialinjected as necessary to produce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In oneembodiment, a therapeutically effective dosage is formulated to containa concentration of at least about 0.1% w/w up to about 90% w/w or more,in certain embodiments more than 1% w/w of the active components of thecomposition to the treated tissue(s).

The components of the composition may be suspended in micronized orother suitable form or may be derivatized to produce a more solubleactive product or to produce a prodrug. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the components of the compositionin the selected carrier or vehicle. The effective concentration issufficient for ameliorating the symptoms of the condition and may beempirically determined.

-   -   C. Lyophilized Powders

Of interest herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They may also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving the componentsof the composition provided herein, or a pharmaceutically acceptablederivative thereof, in a suitable solvent. The solvent may contain anexcipient which improves the stability or other pharmacologicalcomponent of the powder or reconstituted solution, prepared from thepowder. Excipients that may be used include, but are not limited to,dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose,sucrose or other suitable agent. The solvent may also contain a buffer,such as citrate, sodium or potassium phosphate or other such bufferknown to those of skill in the art at, in one embodiment, about neutralpH. Subsequent sterile filtration of the solution followed bylyophilization under standard conditions known to those of skill in theart provides the desired formulation. In one embodiment, the resultingsolution will be apportioned into vials for lyophilization. Each vialwill contain a single dosage or multiple dosages of the components ofthe composition. The lyophilized powder can be stored under appropriateconditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcomponents of the composition. Such amount can be empiricallydetermined.

-   -   D. Topical Administration

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture may be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The components of the composition or pharmaceutically acceptablederivatives thereof may be formulated as aerosols for topicalapplication, such as by inhalation. These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, in one embodiment, havediameters of less than 50 microns, in one embodiment less than 10microns.

The components of the composition may be formulated for local or topicalapplication, such as for topical application to the skin and mucousmembranes, such as in the eye, in the form of gels, creams, and lotionsand for application to the eye or for intracisternal or intraspinalapplication. Topical administration is contemplated for transdermaldelivery and also for administration to the eyes or mucosa, or forinhalation therapies. Nasal solutions of the active components of thecomposition alone or in combination with other pharmaceuticallyacceptable excipients can also be administered.

These solutions, particularly those intended for ophthalmic use, may beformulated as 0.01% 10% (vol %) isotonic solutions, pH about 5 7, withappropriate salts.

-   -   E. Compositions for Other Routes of Administration

Other routes of administration, such as transdermal patches, includingiontophoretic and electrophoretic devices, and rectal administration,are also contemplated herein.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by molding. The weight of a rectal suppository, inone embodiment, is about 2 to 3 gm.

Tablets and capsules for rectal administration are manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration.

-   -   F. Targeted Formulations

The components of the composition provided herein, or pharmaceuticallyacceptable derivatives thereof, may also be formulated to be targeted toa particular tissue, receptor, or other area of the body of the subjectto be treated. Many such targeting methods are well known to those ofskill in the art. All such targeting methods are contemplated herein foruse in the instant compositions.

In one embodiment, liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. Briefly, liposomes such asmultilamellar vesicles (MLV's) may be formed by drying down eggphosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) onthe inside of a flask. A solution of the components of the compositionprovided herein in phosphate buffered saline lacking divalent cations(PBS) is added and the flask shaken until the lipid film is dispersed.The resulting vesicles are washed to remove unencapsulated components ofthe composition, pelleted by centrifugation, and then resuspended inPBS.

Kits of the Invention

According to another aspect of the invention, kits are provided. Kitsaccording to the invention include package(s) comprising compositions ofthe invention.

The phrase “package” means any vessel containing compositions presentedherein. In preferred embodiments, the package can be a box or wrapping.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. Examples of pharmaceuticalpackaging materials include, but are not limited to, blister packs,bottles, tubes, inhalers, pumps, bags, vials, containers, syringes,bottles, and any packaging material suitable for a selected formulationand intended mode of administration and treatment.

The kit can also contain items that are not contained within the packagebut are attached to the outside of the package, for example, pipettes.

Kits may optionally contain instructions for administering compositionsof the present invention to a subject having a condition in need oftreatment. Kits may also comprise instructions for approved uses ofcomponents of the composition herein by regulatory agencies, such as theUnited States Food and Drug Administration. Kits may optionally containlabeling or product inserts for the present compositions. The package(s)and/or any product insert(s) may themselves be approved by regulatoryagencies. The kits can include compositions in the solid phase or in aliquid phase (such as buffers provided) in a package. The kits also caninclude buffers for preparing solutions for conducting the methods, andpipettes for transferring liquids from one container to another.

The kit may optionally also contain one or more other compositions foruse in combination therapies as described herein. In certainembodiments, the package(s) is a container for intravenousadministration. In other embodiments, compositions are provided in aninhaler. In still other embodiments compositions are provided in apolymeric matrix or in the form of a liposome.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

EXAMPLES Example 1

Experimental Procedures

Experimental procedures used to demonstrate presence of BRAF oncogenemutation in rheumatoid arthritis patients and in particular rheumatoidarthritis synovial fibroblasts, transformation of rheumatoid arthritissynovial fibroblast by activating mutation of the BRAF oncogene, andreversal of BRAF-induced transformed rheumatoid arthritis synovialfibroblast phenotype by reducing total BRAF activity, indicating BRAF astarget for therapeutic intervention in patients suffering fromBRAF-induced rheumatoid arthritis.

RA Synovial Fibroblasts.

Synovial tissue was obtained with Institutional Review Board approvalfrom 9 patients with severe RA patients and an 80 y/o woman with severeosteoarthritis (OA) undergoing joint replacement surgery. Patients withRA included 7 women and 2 men ages 30 to 74 years. Synovial tissues weredigested with collagenase, and the resulting cells were filtered throughsterile gauze, washed with Hanks BSS, reconstituted in 90% FCS and 10%DMSO, and stored frozen in liquid nitrogen until used. Synovial cells(5×10⁶-20×10⁶) were thawed at 37° C., immediately diluted 10-fold inDMEM/F12 (1:1) medium, centrifuged and reconstituted in DMEM/F12 mediumcontaining 10% FCS and Hepes buffer. Synovial cells obtained fromsynovial tissues were added to T25 tissue flasks for 2 hours and thenrinsed with Hanks BSS to remove non-adherent cells. Fresh DMEM/F12 (1:1)medium containing 5-10% FCS and Hepes buffer was added to the flasks andthe cells were incubated at 37° C. in the presence of 5% CO₂ for 3 to 14days. RNA was isolated from first passage synovial fibroblasts with theQIAamp RNA Blood Mini Kit (Qiagen, Sylmar, Calif.) according to themanufacturer's protocol.

PCR.

Reverse transcription PCR (RT-PCR) was performed with OneStep RT-PCRKit™ (Qiagen, Valencia Calif.) according to the manufacturer's protocol.The forward primer was: 5′-GGGCCCCGGCTCTCGGTTAT-3′ (SEQ ID NO:3), andthe reverse primer was 5′-TGCTACTCTCCTGAA CTCTCTCACTCA-3′ (SEQ ID NO:4).These primers are located outside of the coding region of BRAF. BRAFsequences, both genomic and mRNA, can be obtained from publicallyaccessible National Center for Biotechnology Information (NCBI;Bethesda, Md.) at the website: www.ncbi.nlm.nih.gov. The design of theprimers used to analyze BRAF mRNA is based on the BRAF mRNA sequenceobtained from NCBI under the NCBI Accession No. NM_(—)004333.4. NestedPCR was done subsequently with primers inside of the coding region. Theforward primer was 5′-GTTCAACGGGGACATGGA-3′ (SEQ ID NO:5) beginning atnucleotide 54 from the ATG start site (exon 1) and the reverse primerwas 5′-ATGGTGCGTTTCCT GTCC-3′ (SEQ ID NO:6) beginning at nucleotide 2279of BRAF. Nested PCR was done with Easy A™ high fidelity cloning enzymeand mastermix (Agilent Technologies, Santa Clara, Calif.) with 1microliter of the RT-PCR product as template. PCR conditions were 1minute of denaturation at 94° C., 1 minute of annealing at 58° C., and 6minutes of extension at 72° C. for 40 cycles. The PCR product waselectrophoresed in 0.8% agarose, and cDNA was isolated from the gel withQIAquick Gel Extraction Kit (Qiagen, Valencia Calif.) according to themanufacturer's protocol. Purified DNA was sequenced by SeqWright,(Houston, Tex.) with the nested primers as well as internal primers.Nested PCR was also done with the “Fast COLD PCR” technique performed asdescribed previously and analyzed by electrophoresis in 1.0% Seakem-1.0%Nusieve agarose gels (FMC BioProducts, Rockland Me.) (Li, J. andMakrigiorgos, G. M. (2009) Biochem Soc Trans, 37, 427-432). “COLD PCR”takes advantage of the lower melting temperatures of some mutant strandsto facilitate their preferential amplification. Primers were designed toamplify a 191 bp fragment centered at V600. The unique restriction siteSfcI was selected to identify mutations in the first nucleotide ofV600R. The forward primer was 5′-ACTGCACAGGGCATG-GAT-3′ (SEQ ID NO:7),and the reverse primer was 5′-TCTGG TGCCATCCACA AAA-3′ (SEQ ID NO:8).

Restriction Enzyme Mapping.

BRAF contains a single SfcI restriction site that includes the firstnucleotide of V600 codon. Complete digestion of BRAF with SfcI indicatesthe presence of wild-type sequence in both PCR amplified alleles.Mutations in nucleotides comprising the SfcI site are expected to resultin partial, or incomplete cutting in the presence of excess enzyme. BRAFwas digested with SfcI for 20 hours at 25° C. with a 10-fold excess ofenzyme required for complete digestion.

siRNA Transfection.

RA fibroblasts were plated at 10-20% confluence overnight in 24-wellplates and transfected in triplicate wells with Human BRAF On-TargetplusSMARTpool Cat# L-003460-00 and On-Target plus Control Pool Non-Targetingpool Cat# D-001810-10-05 (Thermo Scientific Dharmacon, Lafayette, Colo.)with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, Calif.) according tothe manufacturer's protocol for “forward transfection.” Cells werecounted in three separate areas of each well at the time siRNA was addedand again 72 hour later. Data were expressed as mean number of cells inone microscopic field±SD of triplicate determinations. Significantinhibition of cell growth in response to BRAF siRNA compared to controlsiRNA was determined by Students t test. In order to confirmdown-regulation of BRAF in response to BRAF siRNA, fibroblasts treatedwith BRAF siRNA and control siRNA were lysed in 10% SDS and evaluatedfor BRAF and actin by Western blotting to nitrocellulose after SDS-PAGEin 4-20% gradient gels (NuSep, Lawrenceville, Ga.). Nitrocellulose blotswere incubated with rabbit antibodies to BRAF (Abgent, San Diego,Calif.) and goat antibodies to beta-actin (Santa Cruz Biotechnology,Santa Cruz, Calif.) followed by HRP-conjugated polyclonal goatanti-rabbit and rabbit anti-goat secondary antibodies, respectively. Theblots were then developed with SuperSignal West Pico ChemiluminescentSubstrate (Thermo Scientific, Rockford, Ill.) for detection of HRP andimaged with ChemiDoc XRS (BioRad, Hercules, Calif.).

Results

Loss of Fibroblast Contact Inhibition.

Rheumatoid synovial fibroblasts showed absence of contact inhibitionwith a representative example shown (FIG. 1A). Loss of contactinhibition suggests that rheumatoid synovial fibroblasts have undergonetransformation as previously described (Pap, T., et al. (2000) ArthritisRes, 2, 361-367; Karouzakis, E., et al. (2006) Immunol Lett, 106, 8-13;Mercer, K., et al. (2005) Cancer Res, 65, 11493-11500).

Characterization of BRAF Amplified from Synovial Fibroblasts by RT andNested PCR.

RNA was isolated from first passage fibroblasts, reverse transcribed,and cDNA was amplified by PCR with primers outside of the coding region.Nested PCR with primers within the coding region resulted in a DNAfragment of the expected size with aberrant splice variants also evidentin three patients (FIG. 1B). PCR products were subjected toelectrophoresis in 0.8% agarose, and DNA was isolated from the gels.Direct DNA sequencing documented full-length BRAF from all patients aswell as aberrant splice variants of BRAF in RA patients 6, 8, and 9(FIG. 1B). DNA sequences of the splice variants showed the followingexon deletions: RA6: exons 2-15, RA8: exons 3-8, RA9: exons 4-13 (FIG.1C). RA8 splice variant retained the kinase domain but lacked exon 3that contains a portion of the autoinhibitory domain that suppresseskinase activity (Tran, N. H., et al. (2005) J Biol Chem, 280,16244-16253). BRAF splice variants that remove N-terminal autoinhibitorysequences have been associated with enhanced BRAF activity in cancercells (Baitei, E. Y., et al. (2009) J Pathol, 217, 707-715; Tran, N. H.,et al. (2005) J Biol Chem, 280, 16244-16253). Direct DNA sequencing offull-length transcripts with the antisense nested primer showed twoseparate sequences corresponding to residue 600 consistent with bothwild-type and V600R mutation in synovial fibroblasts from patients 1 and6 (FIG. 2). V600R is reported as a gain-of-function mutation with strongenhancement (250-fold) of BRAF activity (Wan, P. T., et al. (2004) Cell,116, 855-867).

Restriction Enzyme Mapping.

In order to confirm the mutations in BRAF cDNA, we performed restrictionmapping of the region that includes V600. SfcI is a unique restrictionsite in BRAF that includes the first nucleotide of V600 codon (FIG. 3A).Incomplete digestion of 2300 bp BRAF cDNA to 1700 bp and 600 bpfragments in the presence of excess enzyme confirms the mutation V600Rin cDNA. Therefore, cDNA from RA6 was digested with a 10-fold excess ofSfcI and examined by gel electrophoresis. Incomplete digestion of the2300 bp PCR product from RA6 was observed, consistent with the V600Rmutation in RA6 cDNA (FIG. 3B). In contrast, SfcI completely digestedthe 2300 bp PCR product from RA3 without mutation of BRAF. In order toconfirm the V600 mutation in RA1, we used “Fast COLD PCR” to enhanceselection of mutant DNA strands as previously described (Li, J. andMakrigiorgos, G. M. (2009) Biochem Soc Trans, 37, 427-432). As shown,digestion of a 191 bp fragment, centered on V600, with an excess of SfcIfailed to completely digest RA1 cDNA, consistent with the V600R mutation(FIG. 3C). In contrast, SfcI completely digested the 191 bp from RA3without mutation of BRAF to produce two fragments, each approximatelyhalf the size of the 191 bp fragment.

BRAF Function in RA Synovial Fibroblasts.

Suppression of BRAF by siRNA inhibited growth of fibroblasts from bothpatients (RA 1 & RA6) with V600R mutations of BRAF and from RA8 with theBRAF splice variant with an intact kinase domain but loss of a portionof the autoinhibitory domain. In contrast, BRAF-specific siRNA did notinhibit growth of synovial fibroblasts from RA2 and RA4 withoutmutations of BRAF. In addition, BRAF siRNA did not inhibit growth offibroblasts from patient OA7 with osteoarthritis (FIG. 4A). BRAF siRNAinhibited the production of BRAF as demonstrated by Western blotting ofsynovial fibroblast lysates from patient RA6 (FIG. 4B).

Discussion

The RAS/RAF/MEK/ERK pathway is referred to as the mitogen-activatedprotein kinase (MAPK) pathway. Activation of MAPK results in cellproliferation, survival, and transformation. BRAF is one of threeisoforms of RAF, but only BRAF is frequently mutated in various cancers(Garnett, M. J. and Marais, R. (2004) Cancer Cell, 6, 313-319). Somemutations in the activation segment of BRAF kinase have gain-of-functionactivity responsible for cellular transformation leading to melanoma andother cancers (Wan, P. T., et al. (2004) Cell, 116, 855-867). Theobservation that mutations of BRAF induce transformation of embryonicfibroblasts, but not several other somatic cells, led us to search forsimilar mutations in RA synovial fibroblasts as a mechanism for theirtransformation (Mercer, K., et al. (2005) Cancer Res, 65, 11493-11500).Residue 600 is most frequently mutated with V600E predominating. V600Roccurs less frequently but is also a strong (250-fold) enhancer of BRAFkinase activity (Wan, P. T., et al. (2004) Cell, 116, 855-867).Identification of V600R gain-of-function mutations of BRAF in RAsynovial fibroblasts in RA patients is consistent with this as apotential mechanism for synovial fibroblast transformation in thepathogenesis of erosive joint disease in some patients. BRAF siRNAinhibited growth of synovial fibroblasts from both patients with BRAFV600R mutations, confirming the functional significance of BRAFmutations on synovial fibroblast growth.

BRAF has two normal isoforms associated with multiple splice variants.However, aberrant splice variants of BRAF were recently reported in bothcolon and thyroid cancers (Baitei, E. Y., et al. (2009) J Pathol, 217,707-715; Seth, R., et al. (2009) Gut, 58, 1234-1241). Some aberrantsplice variants have been shown to activate the MAP kinase signalingpathway suggesting that BRAF splice variants may function as analternative mechanism for oncogenic BRAF activation (Seth, R., et al.(2009) Gut, 58, 1234-1241). Consistent with this idea, a BRAF N-terminalautoinhibitory domain has been identified, and loss of the inhibitorydomain resulted in increased BRAF kinase activity (Lefevre, S., et al.(2009) Nat Med, 15, 1414-1420). Thus, certain aberrant isoforms thatexclude portions of the autoinhibitory domain but retain the kinasedomain may have enhanced kinase activity. In our study, one RA patient(RA8) had an aberrant BRAF splice variant with deletion of exon 3containing a portion of the autoinhibitory domain; however, an intactkinase domain was retained. Proliferation of synovial fibroblasts fromRA8 was inhibited by BRAF-specific siRNA. The finding ofgain-of-function BRAF mutations and splice variants is consistent withmultiple mechanisms involving BRAF that may lead to transformation offibroblasts in RA synovium.

Patients with cancers due to mutations of oncogenes often produceantibodies to altered or cryptic epitopes. Mutations of BRAF occur in15% of cancers with the highest incidence in melanomas where 9% ofpatients have serum antibodies specific for BRAF (Davies, H., et al.(2002) Nature, 417, 949-954; Fensterle, J., et al. (2004) BMC Cancer, 4,62). In a recent study, 9 of 19 patients with rheumatoid arthritis werereported to have serum antibodies to BRAF assayed by Western blot, afinding that is consistent with the presence of an altered BRAF proteinin these patients (Auger, I., et al. (2009) Ann Rheum Dis, 68, 591-594).We did not have matched serum samples to evaluate a correlation betweenthe presence of serum antibodies to BRAF and BRAF mutations or aberrantsplice variants in synovial fibroblasts. Further studies are required toevaluate these associations. It is likely that our study underestimatesthe frequency of BRAF mutations in RA synovial fibroblasts due to thetechnical difficulty inherent in identifying heterozygous mutations. Inaddition, some aberrant splice variants lacking the autoinhibitorydomain would be missed as a result of our selection of PCR primersrequiring the presence of exon 1.

Synovial fibroblasts destroy articular cartilage and bone in rheumatoidarthritis. It is the discovery of the invention that mutations of theBRAF gene can transform synovial fibroblasts responsible for thedestruction of articular cartilage and bone in rheumatoid arthritis andidentifies BRAF gene as a new target for therapeutic intervention.

Transformation of synovial fibroblasts in RA patients by mutant BRAFprotein due to mutations in the BRAF gene include or may include, butare not limited to, loss of synovial fibroblast contact inhibition ofcellular growth, increase in cellular proliferation, ability to grow insoft agar, increase in cellular mobility, ability to migrate andaccumulate at cartilage and/or bone, ability to migrate and accumulateat cartilage and/or bone using in vivo model systems,anchorage-independent growth, morphological changes, focus formation ona monolayer, increase in life span or immortalization, and/ortumorigenicity in nude mice.

Role for mutant BRAF protein in the transformation of synovialfibroblast responsible for destruction of articular cartilage and bonein rheumatoid arthritis is defined by the presence of activating BRAFgene mutations in rheumatoid synovial fibroblasts. Characterization ofthe BRAF gene from rheumatoid synovial fibroblasts from 9 rheumatoidarthritis patients showed mutation of codon 600 of exon 15 changingvaline 600 encoded by GTG to arginine encoded by AGG for RA6 patient andAGA for RA1 patient (FIG. 2). In one publication, valine 600 of BRAF waspreviously designated valine 599 in the 5′ coding region of BRAF(Wellbrock, C., et al. (2004) Nat Rev Mol Cell Biol, 5, 875-885).Presence of this V600R gain-of-function point mutation in BRAF proteinsignificantly activates basal BRAF kinase activity (Wan, P. T., et al.(2004) Cell, 116, 855-867). With increasing sample size beyond 9rheumatoid arthritis patients so far analyzed by us, it is anticipatedthat other codons, CGT, CGC, CGA, and CGG, for arginine may also encodefor BRAF V600R mutation.

In addition to V600R gain-of-function BRAF mutation, other BRAFmutations are anticipated to transform synovial fibroblasts responsiblefor the destruction of articular cartilage and bone in rheumatoidarthritis. Point mutations affecting kinase activity and cellulartransformation have been described for BRAF from a number of humancancers. These gain-of-function point mutations occur primarily in thekinase domain concentrating in and/or around the activation segment, aregion of 10-30 amino acids bounded by almost invariant DFG and APEmotifs, and in the glycine-rich P loop of the N lobe (Wan, P. T., et al.(2004) Cell, 116, 855-867; Davies, H., et al. (2002) Nature, 417,949-954). The P loop interacts with the activation segment to keep BRAFin an inactive conformation (see FIG. 4 of Wan, P. T., et al. (2004)Cell, 116, 855-867). Similar to activated RAS phosphorylation of BRAFthreonine 599 (major activation segment phosphorylation site) and serine602 (minor activation segment phosphorylation site), the mis-sensemutations in P loop or activation segment can activate BRAF bydestabilizing inactive and/or stabilizing active kinase conformation(Wan, P. T., et al. (2004) Cell, 116, 855-867).

The region around BRAF activation segment extends from amino acid 583 to623 of SEQ ID NO:2 with the BRAF activation sequence from amino acid594-623, as is given below, with highly conserved residues found to bemutated in a number of human cancers indicated by bold and doubleunderline (Wan, P. T., et al. (2004) Cell, 116, 855-867; Davies, H., etal. (2002) Nature, 417, 949-954; Garnett, M. J. and Marais, R. (2004)Cancer Cell, 6, 313-319):

594 FLH E DLTVKIG DFGL AT VK SRWSGSHQFEQLSGSILWMAPE 623

Anticipated BRAF mis-sense mutations in or around BRAF activationsegment are expected to transform synovial fibroblasts responsible forthe destruction of articular cartilage and bone in rheumatoid arthritisare: E586K, D587A, D594G, D594K, D594V, F595L, G596R, L597Q, L597R,L597S, L597V, T599I, V600D, V600E, V600G, V600K, K600M, K601E, andK601N. These mutations are based on documented BRAF mis-sense mutationsin human cancers (Wan, P. T., et al. (2004) Cell, 116, 855-867; Davies,H., et al. (2002) Nature, 417, 949-954; Garnett, M. J. and Marais, R.(2004) Cancer Cell, 6, 313-319).

The P loop is part of the N lobe in the crystal structure of BRAF (seeFIG. 4 of Wan, P. T., et al. (2004) Cell, 116, 855-867) and extends fromamino acid 462-469 of SEQ ID NO:2 containing a highly conservedglycine-rich motif, GXGXXG motif (see FIG. 2b of Davies, H., et al.(2002) Nature, 417, 949-954), with the sequence given below and residuesfound to be mutated in a number of human cancers indicated by bold anddouble underline:

462 RI G S G S FG 469

The BRAF mis-sense mutations in P loop that are expected to transformsynovial fibroblasts responsible for the destruction of articularcartilage and bone in rheumatoid arthritis are: R462I, I463S, G464E,G464R, G464V, G466A, G466E, G466R, G466V, F468C, G469A, G469E, G469R,G469S, and G469V.

Additional mutations outside of the activation segment region and P loopbut within the kinase domain found in human cancers are: V459L, K475E,N581S, R682Q, and A728V (Wan, P. T., et al. (2004) Cell, 116, 855-867;Davies, H., et al. (2002) Nature, 417, 949-954; Garnett, M. J. andMarais, R. (2004) Cancer Cell, 6, 313-319). Other BRAF mis-sensemutations are: M117R, I326T, K439Q, K439T, T440P, and V459L (Wan, P. T.,et al. (2004) Cell, 116, 855-867; Davies, H., et al. (2002) Nature, 417,949-954; Garnett, M. J. and Marais, R. (2004) Cancer Cell, 6, 313-319).Furthermore, a more extensive listing of BRAF mutations in human cancerscan be found at the website (http://ww.sanger.ac.uk/genetics/CGP/cosmic)using the search term BRAF. While this listing is not meant to beexhaustive, these BRAF mis-sense mutations within the context ofsynovial fibroblasts are anticipated to transform the fibroblastsresponsible for the destruction of articular cartilage and bone inrheumatoid arthritis.

It is noted that V600E mis-sense mutation may account for at least 80%of BRAF mutations in human cancers (Wan, P. T., et al. (2004) Cell, 116,855-867; Davies, H., et al. (2002) Nature, 417, 949-954; Garnett, M. J.and Marais, R. (2004) Cancer Cell, 6, 313-319).

In addition to the mis-sense, gain-of-function mutations, theRAS-RAF-MEK-ERK signal transduction pathway can become altered by thepresence of internally truncated BRAF proteins, resulting intransformation of rheumatoid synovial fibroblasts. Aberrantly splicedBRAF transcripts were found in some rheumatoid synovial fibroblasts,resulting in loss of BRAF coding sequences corresponding to exons 2-15,exons 2-16, exons 3-8, exons 4-13, and exons 4-16. In the case of exon3-8 deletion, in which the auto-inhibitory domain of BRAF is deletedwhile the kinase domain remains intact, the internally truncated BRAFprotein promoted cellular growth of the affected rheumatoid synovialfibroblasts. The constitutively active BRAF kinase phosphorylates andactivates MEK1/2 proteins, ultimately resulting in increasedproliferation of synovial fibroblast cells. Analysis of rheumatoidsynovial fibroblasts from additional rheumatoid arthritis patients willprovide more examples of other aberrantly spliced BRAF transcriptsdiffering in the exons deleted from the mRNA transcript.

Finding aberrantly spliced transcripts for BRAF in rheumatoid synovialfibroblasts is significant and these, like the mis-sense BRAF mutanttranscripts, are also therapeutic targets in the treatment of rheumatoidarthritis. Internally truncated BRAF proteins, e.g. in which exons 4-15or 2-17 are deleted, are reported to promote cellular transformation,mediated perhaps through activation of the RAS-RAF-MEK-ERK/MAP kinasesignaling pathway leading to increased phosphorylation and activation ofERK kinase protein (Baitei, E. Y., et al. (2009) J Pathol, 217,707-715). In the case of exon 2-17 deletion, it is noteworthy that boththe auto-inhibitory domain and almost the entire kinase domain aremissing in the truncated protein. Yet, this truncated protein, obtainedfrom translation of exon 1 and 18, can promote cellular transformation.It is anticipated that truncated, internally deleted BRAF proteins fromaberrantly spliced BRAF transcripts will similarly transform synovialfibroblast. It is significant that a number of aberrantly spliced BRAFtranscripts were present in rheumatoid synovial fibroblast, and theseaberrant transcripts and associated proteins serve as therapeutictargets in rheumatoid arthritis.

In addition to mis-sense mutation and aberrant RNA splicing, mutant BRAFgene and protein with internal deletion(s) and/or insertion(s) areanticipated. Such changes in rheumatoid synovial fibroblast can lead totransformation of synovial fibroblast, and as such, these BRAF genes andproteins are therapeutic target in rheumatoid arthritis.

It is noted that in addition to the BRAF coding sequence and proteinsequence shown in SEQ ID NO:1, additional isoforms of BRAF exist. Forexample, an isoform of BRAF is due to alternative splicing of exon 8bbetween exons 8 and 9 (encoding “EKFPEVELQDNR” which when present isinserted, e.g., between aspartic acid 380 and aspartic acid 381 of SEQID NO:2) and/or alternative splicing of exon 9b between exons and 10 ofSEQ ID NO:1 (encoding “APLNQLMRCLRKYQSRTPSPLLHSVPSEIVFDFEPGPVFR” whichwhen present is inserted between glycine 392 and glycine 393 of SEQ IDNO:2) (Hmitou, I., et al. (2007) Mol Cell Biol, 27, 31-43). The fourdifferent BRAF isoforms are differentially regulated which in turnaffects downstream MEK activation (Hmitou, I., et al. (2007) Mol CellBiol, 27, 31-43). It is expected that these BRAF isoforms will bepresent in rheumatoid synovial fibroblasts. In addition, BRAF mutations,described above, may be present in BRAF isoforms, and as such, theseBRAF isoforms and their mutants are potential therapeutic targets inrheumatoid arthritis.

Discovery that BRAF mutations are found in rheumatoid synovialfibroblast from rheumatoid arthritis patients provides a therapeutictarget in the treatment of rheumatoid arthritis. Presence of BRAFmutations transform rheumatoid synovial fibroblast, and to assess theinvolvement of BRAF in transformation of rheumatoid synovial fibroblast,a method is described in which the activated BRAF mutant proteinexpression is decreased through the use of e.g. siRNA (small interferingRNA) directed against BRAF sequences and the treated cells assessed fortransformed phenotype, such as contact inhibition and/or cellularproliferation. Unlike wild-type BRAF protein, loss of mutant BRAFprotein expression reverses the transformed synovial fibroblastphenotype, indicating that a subject is suffering from a rheumatoidarthritis associated with the BRAF oncogene. Protocols for the isolationof synovial fibroblasts, cell culture condition of the isolated primarysynovial fibroblast, treatment with siRNA, and assays for contactinhibition and cell proliferation are provided.

Double stranded RNA(s) can suppress expression of a specific gene ofinterest in a homology-dependent manner in a process called “RNAinterference” or “RNAi” by targeting mRNA molecules for degradation(Sharp, P. A. and Zamore, P. D. (2000) Science, 287, 2431-2433; Bosher,J. M. and Labouesse, M. (2000) Nat Cell Biol, 2, E31-36). SiRNAs aresmall interfering RNAs which are double-stranded RNA molecules withapproximately 20-25 nucleotides in length that participate in the RNAipathway. Typically, siRNAs have a short double-stranded RNA region(usually 21 bp) with a 2-nucleotide overhang. A number of commercialcompanies (such as Thermo Fisher Dharmacon and OriGene) supply eitherunmodified or modified siRNAs designed to knockdown BRAF transcriptlevels and consequently BRAF protein expression.

To obtain long term down-regulation of BRAF expression, siRNA expressionvector may be introduced into synovial fibroblast cells with a loopbetween two strands homologous to BRAF sequences to produce shorthairpin RNA (shRNA) produced by RNA polymerase III transcription usingRNA polymerase III promoter, such as U6 or H1. In addition, promotersfor shRNA genes, such as U1, may be used to produce shRNAs. The shRNAsare processed to produce siRNAs for down-regulation of BRAF transcriptlevel and consequently BRAF protein level. Design and production ofsiRNAs and shRNAs toward BRAF are discussed in U.S. patent applicationSer. No. 10/834,665 entitled “Treatment of Cancer by Inhibiting BRAFExpression,” filed on Jun. 3, 2003 and U.S. patent application Ser. No.11/270,796 entitled “Treatment of Cancer by Simultaneous Inhibition ofBRAF and Restoration or Mimicry of p16^(INK4A) Activity,” filed on Nov.8, 2005 and BRAF shRNA expression constructs may be obtained fromcommercial sources, such as GeneCopoeia, OriGene, and QiagenSABiosciences.

In addition to siRNAs and shRNAs, BRAF expression may be down-regulatedby the use of precursor microRNAs (miRNAs) expression vector used toproduce precursor miRNAs and subsequent processing to miRNAs. MaturemiRNAs usually directed to the 3′ untranslated region (3′ UTR)down-regulates gene expression either by mRNA degradation or interferingwith translation (Bartel, D. P. (2009) Cell, 136, 215-233). The miRNAsfor BRAF may be obtained from commercial sources, such as GeneCopoeiaand OriGene. Thus, siRNA, shRNA, and/or miRNA to BRAF can be used todown-regulated BRAF expression and determine if BRAF gene is associatedwith transformation of rheumatoid synovial fibroblasts in rheumatoidarthritis, thereby establishing whether a subject is suffering from arheumatoid arthritis associated with the BRAF oncogene.

Other methods based on homology to the BRAF nucleic acid sequence couldbe used to downregulate BRAF expression and these would also include useof anti-sense nucleic acid approaches, including but not limited toshort anti-sense oligonucleotides (DNA or RNA) or long anti-sense RNAtranscripts, ribozymes, etc. It should also be noted that the introducednucleic acid to downregulate BRAF expression by targeting BRAF nucleicacid sequences may be unmodified RNA or alternatively chemicallymodified (e.g., incorporation of 2′-O-methyl or 2′-O-methoxyethyl-group)so as to increase efficiency of delivery, reduce non-specificdegradation by nucleases, increase efficiency of targeting BRAFsequences, reduce off-target effects, etc.

BRAF inhibitors may be used to assess if a subject is suffering from arheumatoid arthritis associated with the BRAF oncogene. Use of BRAFinhibitors in the absence of activated RAS is expected to inhibit cellproliferation and promote cell death (19). However, its use in activatedRAS cells results in MEK-ERK activation by promoting dimerization ofCRAF, one of three RAF isoforms, with wild-type BRAF or kinase-deadBRAF, or another CRAF monomer which are kinase-active dimers (Cichowski,K. and Janne, P. A. (2010) Nature, 464, 358-359). Thus, by knowing thestatus of RAS, it is anticipated that BRAF inhibitors may be used in theabsence of RAS mutation to determine if a subject is suffering from arheumatoid arthritis associated with the BRAF oncogene.

Using art known to a skilled professional, mutation in the BRAF sequencecan be determined by extracting RNA from synovial fibroblast, RT-PCR,and nested PCR with BRAF primers to obtain BRAF cDNA which can befurther subjected to sequence determination and/or to restriction enzymedigestion analysis with SfcI restriction enzyme. BRAF V600R mutation canbe identified on the basis of sequence and its presence is consistentwith loss of SfcI cleavage at codon 600. While electrophoretic analysisof PCR products can show presence of aberrantly spliced transcripts,sequence determination can be used to definitely demonstrate the natureof the aberrantly spliced transcripts. Mutations in the BRAF nucleicacid may also be directly determined by analyzing BRAF genomic DNA withPCR, DNA sequence determination, and restriction enzyme analysis.Standard molecular biology techniques may be found in Ausubel, F. M.(2002) Short protocols in molecular biology: a compendium of methodsfrom Current protocols in molecular biology. 5th ed. Wiley, New York;Ausubel, F. M. (1987) Current protocols in molecular biology. Publishedby Greene Pub. Associates and Wiley-Interscience: J. Wiley, New York;and Sambrook, J. and Russell, D. W. (2001) Molecular cloning: alaboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

Finally, by establishing BRAF as a therapeutic target in the treatmentof rheumatoid arthritis, the current invention provides a rationale fortreatment of rheumatoid arthritis patients. BRAF or RAF inhibitors,which may work by binding directly to BRAF, may be used to reversesynovial fibroblast transformation dependent on BRAF mutant protein ortruncated BRAF protein. Use of specific inhibitors to treat rheumatoidarthritis should also take RAS status into account. Examples of BRAF orRAF inhibitors include but are not limited to: PLX4032 (Plexxikon, Inc.,Berkeley, Calif.; (Tap, W. D., et al. (2010) Neoplasia, 12, 637-649)),RAF265 (CHIR-26; Novartis Pharmaceuticals, Basel, Switzerland),Sorafenib (BAY43-9006; Bayer, Pittsburgh, Pa.), XL281 (Exelixis, SanFrancisco, Calif.), SB-590885 (SmithKline Beecham, Philadelphia, Pa.),and PLX4720 (Plexxikon, Inc., Berkeley, Calif.). In addition, since MEKand ERK are downstream targets of RAF, compounds that directly bind toMEK or ERK kinases may serve as MEK or ERK inhibitors, respectively(Wong, K. K. (2009) Recent Pat Anticancer Drug Discov, 4, 28-35), andsuch compounds may also be beneficial in treating rheumatoid arthritispatients. Such MEK inhibitors include AZD6244 (ARRY-142886; AstraZeneca,London, England), PD0325901, CI-1040, and XL518 (Exelixis, SanFrancisco, Calif.). In addition, ERK inhibitors include FR180204(EMD4Biosciences, Merck KGaA, Darmstadt, Germany), CAY10561 and 328006(Merck KGaA, Darmstadt, Germany). The list above is by no meansexhaustive and the compounds listed above are for illustrative purposesonly.

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What is claimed is:
 1. A method of identifying a molecule that binds orblocks a BRAF protein in a synovial fibroblast associated with a BRAFoncogene comprising: a. contacting a molecule of interest with the BRAFprotein, and b. determining whether the molecule of interest alters BRAFprotein activity in the fibroblast, alteration of the BRAF proteinactivity being indicative that the molecule of interest binds or blocksthe BRAF protein in the fibroblast.